The basic hypothesis of this proposal is that the ability of alpha adrenergic agonists to stimulate the Na+ pump of vascular smooth muscle may be related to the activation of Na+ influx and/or intracellular Ca++ release which are alpha1-mediated events or the influx of extracellular Ca++ which is an alpha2-mediated event. The project will be aimed at investigating this hypothesis in both quiescent and proliferating cultured vascular smooth muscle cells. These culture systems may serve as a model of normal, differentiated vascular cells (quiescent) or as a model of certain disease states such as atherosclerosis or hypertension in which cells are proliferating. Cells maintained under quiescent or proliferating conditions will be characterized with respect to Na+ pump number by quantitating [3H]-ouabain binding, turnover rate of the Na+ pump by measuring ouabain-sensitive 86Rb uptake and numbers of alpha1 and alpha2 receptors by [3H]-prazosin and [3H]-yohimbine binding. After control values for each of these parameters have been determined, the short-term effects of selective alpha1 and alpha2 agonists and antagonists on Na+ pump rate will be monitored. The role of a particular receptor subtype or subtypes will be assesed by comparison of relative potencies of selective agonists and pA2 values of selective antagonists. A primary effect of alpha agonists in all cells possessing alpha receptors is the mobilization of extracellular or intracellular Ca++. Experiments will be performed to determine the role of Ca++ in alpha-mediated stimulation of the Na+ pump by manipulating these pools of Ca++ and observing the effects on alpha-mediated pump stimulation. An alternative mechanism may be an alpha-stimulated Na+ influx related to the activation of a membrane Na+-H+ exchanger. This possibility will be investigated by measuring changes in [Na+]i by flame photometry and [pH]i by uptake of the weak acid [14C]-DMO in the presence of selective alpha agonists and antagonists. It is also planned to assess the effect on Na+ pump parameters of long term incubations of vascular smooth muscle cells with alpha agonists. In addition, alpha receptor number will also be monitored. It is expected that significant data will be obtained regarding the control of active and passive ion movements in vascular smooth muscle. In view of the role that disturbances of ionic regulation are believed to play in hypertension, it is anticipated that these studies may contribute to the understanding of this disease process.